Abstract Deep vein thrombosis (DVT) is a major public health problem, affecting 600,000 Americans annually with a healthcare cost of $10 billion and more than 100,000 deaths. The American Heart Association recommends catheter-directed thrombolytics or mechanical interventions for critical obstructions to prevent amputation of the afflicted limb or death. These treatment regimens are not effective for the chronic thrombus components present in 27 to 43% of DVT cases. Histotripsy is a novel form of therapeutic ultrasound that employs the mechanical action of bubble clouds to ablate tissue and induce fluid mixing. The scientific premise of this study is histotripsy, a therapeutic ultrasound modality that employs the mechanical action of bubble clouds, induces vigorous fluid mixing for improved thrombolytic delivery and liquefaction of chronic, well organized thrombi. Our preliminary studies demonstrate synergy between histotripsy and the thrombolytic recombinant tissue plasminogen activator (rt-PA) that enhances thrombolysis of a retracted venous clot in vitro. We have also shown histotripsy ablation occurs only after a threshold amount of bubble cloud activity. These preliminary data strongly support our central hypothesis that the mechanical action of histotripsy-induced bubble clouds enhances a catheter-directed thrombolytic. In particular, histotripsy causes ablation of chronic thrombus through bubble cloud formation, and increased rt-PA efficacy via bubble cloud-enhanced fluid mixing. Furthermore, catheter-infusion of thrombus-targeted echogenic drug delivery vesicles will improve thrombolysis through localized rt-PA delivery and thrombus ablation. We will examine this hypothesis through the following three specific aims: In Specific Aim 1, we will develop a prototype DVT histotripsy system to facilitate simultaneous 1-MHz histotripsy exposure, therapy image guidance, and treatment progress monitoring. In Specific Aim 2, we will determine the conditions for histotripsy-enhanced thrombolysis. These data will quantify the synergistic effects observed between the histotripsy excitation and thrombolytic drugs for clot lysis. In Specific 3, recanalization of DVT with histotripsy and thrombolytic will be assessed in vivo. Successful completion of the proposed studies will elucidate the efficacy and potential risks of histotripsy for venous thrombosis, and provide new information to assist the development of targeted ablation methods to improve thrombolysis in the treatment of currently intractable deep vein thrombosis.